A multicenter, randomized, double-blind, controlled study to evaluate the efficacy and safety of dantrolene on ventricular arrhythmia as well as mortality and morbidity in patients with chronic heart failure (SHO-IN trial): rationale and design.

BACKGROUND: Leakage of Ca2+ from the sarcoplasmic reticulum (SR) is a critical contributing factor to heart failure pathophysiology. Therefore, reducing SR Ca2+ leaks may provide significant additive benefits when used in combination with conventional therapies. Dantrolene, a drug routinely used to treat malignant hyperthermia, also stabilizes the cardiac isoform of the release channel (RyR2), thus decreasing SR Ca2+ leaks. The purpose of this study is to evaluate the effect of chronic administration of dantrolene on heart failure and lethal arrhythmia in patients with chronic heart failure and reduced ejection fraction in a multicenter, randomized, double-blind, controlled study. METHODS: Patients with chronic heart failure who had functional status of New York Heart Association class II and III and a left ventricular ejection fraction <40% were treated according to the Japanese Circulation Society, the European Society of Cardiology, and the American Heart Association/the American College of Cardiology guidelines for diagnosis and treatment of acute and chronic heart failure. Patients were randomized and divided into two groups in a double-blind fashion: dantrolene group and placebo group (target sample size: 300 cases). These drugs were administered for 96 weeks. The primary endpoint is cardiovascular death, first hospitalization for exacerbation of heart failure, or lethal arrhythmia [ventricular tachycardia (VT) storm, sustained VT, ventricular fibrillation] for 2 years after starting administration of dantrolene 1 cap (25mg) three times daily (if not tolerable, two times daily) or matching placebo. RESULTS: This paper presents the rationale and trial design of the study. Recruitment for the study started on 8 December 2017. CONCLUSIONS: The results of this trial will clarify the efficacy and safety of dantrolene for ventricular arrhythmia, as well as mortality and morbidity in patients with chronic heart failure and reduced ejection fraction during guideline-directed medical treatment.

BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination.

Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5' to 3' strand resection (DSB resection), with nucleases generating the 3' single-strand DNA (3'ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.

Exon 3 deletion of RYR2 encoding cardiac ryanodine receptor is associated with left ventricular non-compaction.

AIMS: Ryanodine receptor gene (RYR2) mutations are well known to cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, RYR2 exon 3 deletion has been identified in patients with dilated cardiomyopathy (DCM) and/or CPVT. This study aimed to screen for the RYR2 exon 3 deletion in CPVT probands, characterize its clinical pathology, and confirm the genomic rearrangement. METHODS AND RESULTS: Our cohort consisted of 24 CPVT probands. Polymerase chain reaction (PCR)-based conventional genetic analysis did not identify any mutations in coding exons of RYR2 in these probands. They were screened using multiplex ligation-dependent probe amplification (MLPA). In probands identified with RYR2 exon 3 deletion, the precise location of the deletion was identified by quantitative PCR and direct sequencing methods. We identified two CPVT probands from unrelated families who harboured a large deletion including exon 3. The probands were 9- and 17-year-old girls. Both probands had a history of syncope related to emotional stress or exercise, exhibited bradycardia, and were diagnosed with left ventricular non-compaction (LVNC). We examined 10 family members and identified six more RYR2 exon 3 deletion carriers. In total, there were eight carriers, of which seven were diagnosed with LVNC (87.5%). Two carriers under the age of 4 years remained asymptomatic, although they were diagnosed with LVNC. Using quantitative PCR and direct sequencing, we confirmed that the deletions were 1.1 and 37.7 kb in length. CONCLUSION: RYR2 exon 3 deletion is frequently associated with LVNC. Therefore, detection of the deletion offers a new modality for predicting the prognosis of patients with LVNC with ventricular/atrial arrhythmias, particularly in children.

Interference in DNA replication can cause mitotic chromosomal breakage unassociated with double-strand breaks.

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/)KU70(-/-) DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/)LIG4(-/-) Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.